Alzheimer's disease (AD) causes progressive dementia with consequent formation of amyloid plaques, neurofibrillary tangles, gliosis and neuronal loss. The disease occurs in both genetic and sporadic forms whose clinical course and pathological features are quite similar. Three genes have been discovered to date which, when mutated, cause an autosomal dominant form of Alzheimer's disease. These encode the amyloid protein precursor (APP) and two proteins, presenilin-1 (PS1) and presenilin-2 (PS2), which are structurally and functionally related. Mutations in any of the three proteins have been observed to enhance proteolytic processing of APP via an intracellular pathway that produces amyloid beta peptide (Aβ peptide, sometimes referred to as Abeta), a 40–42 amino acid peptide that is the primary component of amyloid plaque in AD (Younkin, Brain Pathol. 1(4):253–62, 1991; Haass, J. Neurosci. 11(12):3783–93, 1991).
Dysregulation of intracellular pathways for proteolytic processing may be central to the pathophysiology of AD. In the case of plaque formation, mutations in APP, PS1 or PS2 consistently alter the proteolytic processing of APP so as to enhance formation of Aβ 1–42, a form of the Aβ peptide which seems to be particularly amyloidogenic, and thus very important in AD. APP localizes to the secretory membrane structure including the cell surface, and has a single C-terminal transmembrane domain. Examples of specific isotypes of APP which are currently known to exist in humans include the 695-amino acid polypeptide described by Kang et. al. (1987), Nature 325: 733–736 which is designated as the “normal” APP; the 751 amino acid polypeptide described by Ponte et al. (1988), Nature 331: 525–527 (1988) and Tanzi et al. (1988), Nature 331: 528–530; and the 770 amino acid polypeptide described by Kitaguchi et. al., Nature 331: 530–532 (1988).
The Aβ peptide is derived from a region of APP adjacent to and containing a portion of the transmembrane domain. Normally, processing of APP at the α-secretase site cleaves the midregion of the Aβ sequence adjacent to the membrane and releases the soluble, extracellular domain of APP from the cell surface. This α-secretase APP processing creates soluble APP-α, which is not thought to contribute to AD. However, pathological processing of APP at the β- and γ-secretase sites, which are located N-terminal and C-terminal to the α-secretase site, releases the Aβ peptide. Processing at the β- and γ-secretase sites can occur in both the endoplasmic reticulum (in neurons) and in the endosomal/lysosomal pathway after re-internalization of cell surface APP (in all cells). The β-secretase cleavage site is located 28 residues from the plasma membrane luminal surface and the γ-secretase cleavage site is located in the transmembrane region. The in vivo processing of the β-secretase site is thought to be the rate limiting step in Aβ production (Sinha and Lieberburg, Proc. Nat'l. Acad. Sci., USA 96(4), 11049–11053, 1999) and as such is a favored target therapeutic target.
Recently, several groups of investigators have reported that a human aspartyl protease (Hu-Asp2) has an activity responsible for the processing of APP at the β-secretase cleavage site. Hu-Asp2 is a membrane-bound aspartyl protease (Yan et al., Nature 402:533–536, 1999; Lin et al., Proc. Nat'l Acad. Sci., USA 97(4): 1456–1460, 2000; Vassar et al., Science, 286:735–741, 1999). Aspartyl proteases such cathepsin D, Pepsin, renin, and viral aspartyl proteases comprise signature catalytic triplets . . . Asp-Thr-Gly . . . and . . . Asp-Ser-Gly . . . . In Hu-Asp2, these signature catalytic triplets are harbored opposite to each other in the bilobed structure of Hu-Asp2. An important feature of Hu-Asp2 that is absent from other known mammalian aspartyl proteases is a stretch of 27 amino acids located near the C-terminus that anchors Hu-Asp2 to the membrane and is essential for the enzyme function in cells.
To date, in vitro assays with recombinant Hu-Asp2 or Hu-Asp2 purified from brain have relied on peptide substrates comprising the β-secretase recognition sequence of the β-amyloid precursor sequence (i.e., APP sequence), such as the APP Swedish mutation sequence . . . EVNL-DAEFR . . . (SEQ ID NO:113) in which the hyphen denotes the point at which the peptide is cleaved by the β-secretase. The same approach has been used for β-secretase assays in cell lines expressing Hu-Asp2.
There presently exists a need to identify compounds that may act as surrogates for the APP substrate of Hu-Asp2. Identifying such substrates facilitates new in vitro and model in vivo assays for identifying therapeutic agents that affect APP processing at the β-secretase site. Ultimately, the identification of such substrates and assays will lead to advances in the identification of therapeutic compounds for the beneficial intervention of Alzheimer's Disease.